1. Field of the Invention
The invention relates to a process for preparing N.sup.6 -substituted NAD, NADP or FAD by Dimroth rearrangement.
2. Brief Description of the Prior Art
NAD(H) or NADP(H) bound to solid polymeric supports or to water-soluble macromolecules have already been under investigation and in use for about 15 years. The coenzymes bound to solid supports are successfully used in affinity chromatography (1). Water-soluble macromolecular NAD(H) or NADP(H) is used as enyzmatically regeneratable coenzyme derivative in enzyme membrane reactors for the continuous preparation of fine chemicals by coenzyme-dependent enzymatic catalysis (2).
A conventional strategy for synthesis of these macromolecular NAD(H) or NADP(H) derivates is: alkylation of the N(1) position of the adenine ring system of NAD or NADP, chemical reduction to N(1)-alkylated NADH or NADPH, Dimroth rearrangement in the basic medium and at elevated temperatures (pH 10-11, 60.degree.-70.degree. C.) to N.sup.6 -alkylated NADH or NADPH, covalent binding of said reduced coenzyme to the macromolecule or enzymatic oxidation to the N.sup.6 -alkylated NAD or NADP and subsequent covalent linking to the macromolecule.
For NAD and NADP the reaction sequence has been employed in the case of the alkylation agents such as iodoacetic acid (3,4), propiolactone (5), 3,4-epoxy butyric acid (6,7) or ethyleneimine (8,9). Coenzymes alkylated in this manner have for the covalent bonding to macromolecules carboxyl or amino groups at the end of the chain at the N.sup.6 -atom of the adenine ring.
For the linking insoluble macromolecules (solid supports, matrices) or soluble, in particular water-soluble, macromolecules are possible Which have one or more functional groups active for the linking. The macromolecules may per se have such functional groups or the functional groups can be introduced into the macromolecules by methods known to the expert. Examples of macromolecules which can be used are: dextrans, polyethylene glycols, polyethyleneimines, polyacrylamides, copolymers such as methylvinyl ether/maleic anhydride, ethylene/maleic anhydride, divinyl ether/maleic anhydride, agarose, glass, cellulose, silica gel, and derivatives of the macromolecules. For the synthesis of macromolecular coenzyme derivatives alternative strategies have been developed which can be considered as variants of the conventional strategy and are intended in many cases to simplify the synthesis. Thus, a covalent coupling has been described which is catalyzed by transglutaminase in the case of binding the conventionally synthesized N.sup.6 -[(6-aminohexyl)carbamoylmethyl[-NAD.sup.+ to water-soluble proteins, for example casein via .gamma.-carboxyamide groups of the L glutamine (10).
It is also possible to prepare firstly N.sup.6 -vinyl derivatives of NAD in conventional manner and to copolymerize them with other vinyl monomers to give macromolecular NAD (5). A simplified copolymerization method (preparation N(1)-vinyl-NAD.sup.+ derivative and simultaneous copolymerization and Dimroth rearrangement) gave a macromolecular NAD derivative which could only be reduced enzymatically to a limited (&lt;40%) extent (11). Based on water-soluble polymers with epoxy groups a method is known in which proceeding from unmodified NAD the N(1)-alkylation, reduction to N(1)-alkylated NADH and Dimroth rearrangement to N.sup.6 -alkylated NADH in covalently bound state at the polymer was carried out in a three-step method (12). Proceeding from NADH even a one-step method is possible because coupling by N(1)-alkylation and Dimroth rearrangement in the basic medium can take place simultaneously under the same conditions. In the best macromolecular NADH was obtained which was oxidizable enzymatically to about 60%.
These simplified methods have the disadvantage that not very well defined macromolecular NAD(H) derivatives arise due to secondary reactions at the coenzyme which are the main cause of the limited oxidizability or reducibility.